Severe acute respiratory syndrome coronavirus (SARS-CoV) is an emerging pathogen responsible for considerable morbidity and mortality worldwide, yet little is known regarding the molecular basis for its pathogenesis. The most significant organ system affected in fatal SARS cases is the respiratory tract. The cynomolgus macaque model best replicates the pulmonary pathogenesis associated with human SARS and was used to prove that SARS-CoV is the primary etiologic agent of SARS, and that type-1 and -2 pneumocytes may be important target cells for SARS-CoV. Our overall goal is to increase our knowledge of SARS-CoV pulmonary pathogenesis. In Specific Aim 1 we will evaluate the pathogenesis of SARS lung disease expanding upon the macaque model. We intend to refine identification of 'target cells permissive for SARS-CoV and possible changes in their distribution at different times after infection. The role of age and immunosuppression, important host factors that increase the severity SARS, will be examined by comparing the development of SARS in macaques of different ages, or treated with different levels of corticosteroids. We will build on the macaque model to include the use of infectious molecularly cloned wild-type SARS-CoV and SARS-CoV containing mutations in specific accessory genes that may contribute to virulence. Specific Aim 2 will focus on the establishment of an appropriate cell culture system in which to evaluate the cellular response to wild-type and engineered SARS-CoV. This will include studies with human pneumocytes and small airway epithelial cells. We also will attempt to infect lung A549 cells and macaques primary macrophages and dendritic cells. Finally, Specific Aim 3 will determine molecular mechanisms and pathogenic signatures of SARS using global gene expression analysis and functional genomics. We will use oligonucleotide microarrays and sophisticated information technologies to examine gene expression changes in response to SARS-CoV infection. Experiments will be performed using RNA isolated from in vitro infections and from cells and tissues from experimentally infected macaques. Collectively, data obtained from the macaque model, cell culture systems, and gene expression analyses should provide new insights into the pathogenesis of SARS, aid in the development of diagnostic and prognostic assays, and identify novel targets for preventive or therapeutic intervention.